The first dual ChE/FAAH inhibitors: New perspectives for Alzheimer's disease?

Article abstract of DOI:10.1021/ml200313p

The treatment of Alzheimer's disease (AD) still remains an area of significant unmet need, with drugs that only target the symptoms of the disease. Therefore, there is considerable need for disease-modifying therapies. The complex etiology of AD prompts s

Full text of DOI:10.1021/ml200313p

Letter
pubs.acs.org/acsmedchemlett
The First Dual ChE/FAAH Inhibitors: New Perspectives for Alzheimer's
Disease?
Angela Rampa,*,† Manuela Bartolini,† Alessandra Bisi,† Federica Belluti,† Silvia Gobbi,†
Vincenza Andrisano,† Alessia Ligresti,‡ and Vincenzo Di Marzo*,‡
†Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
‡Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council, Via Campi Flegrei 34
Comprensorio Olivetti, 80078 Pozzuoli, NA, Italy
S
* Supporting Information
ABSTRACT: The treatment of Alzheimer's disease (AD) still
remains an area of significant unmet need, with drugs that only
target the symptoms of the disease. Therefore, there is
considerable need for disease-modifying therapies. The
complex etiology of AD prompts scientists to develop
multitarget strategies to combat causes and symptoms. To
this aim, we designed, synthesized, and tested four new
carbamates as dual cholinesterase-FAAH inhibitors. The dual activity of these compounds could lead to a potentially more
effective treatment for the counteraction of AD progression, because they would allow regulation of both ACh and eCB signaling
and improve neuronal transmission and/or counteract neuroinflammation.
KEYWORDS: Alzheimer's disease, drug design, FAAH, AChE, BuChE, carbamate inhibitors
lzheimer's disease (AD) is the most common neuro-
degenerative disorder, and its prevalence is increasing
of AD, the modulation of a single factor might not be sufficient
to produce the desired efficacy. Researchers are now turning to
the design of structures that could be able to simultaneously
interact with different targets involved in the pathogenic
process.7,8
Recent advances in the field of the central nervous system
(CNS) strongly suggest that glia (astroglia and microglia) play
an important role in neurodegenerative diseases. Specifically,
microglia, the resident macrophages in the brain, are activated
in response to both Aβ and neuronal damage.9,10
Besides, there is an emerging interest in the relevance of the
endocannabinoid system (eCB) to AD.11,12 The eCB system
consists of lipid signaling molecules that bind to two G-protein-
coupled receptors, named CB1 and CB2: CB1 receptors are
mainly expressed in neurons, while CB2 receptors can be found
in a variety of immune cells, including activated microglia in the
AD brain, most probably as a function of their inflammatory
phenotype.13 The selective expression of CB2 receptors in
regions of neuritic plaques suggests that this receptor plays a
role in controlling the inflammation associated with AD.
Specifically, CB2 receptor expression may be an adaptive
response to excessive inflammation induced in regions of Aβ
deposition aimed at reducing microglia and astrocyte activation.
Additionally, in regions of Aβ-enriched neuritic plaques, an
increased activity of the enzyme fatty acid amide hydrolase
(FAAH) was selectively demonstrated.13 FAAH is an integral
A
together with life expectancy. Although the etiology of AD is
not completely known, histopathological hallmarks such as
amyloid β (Aβ) deposits,1 τ protein aggregation,2 oxidative
stress, inflammation, and dysfunction of acetylcholine (ACh)
signaling in the basal forebrain seem to play significant roles.
Indeed, the evidence that a selective loss of presynaptic
cholinergic neurons occurred in AD patients led in the last
decades to the development of cholinesterase inhibitors
(ChEI),3,4 which temporarily increase the amount of ACh in
the neuronal synaptic cleft by inhibiting acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE), the enzymes
responsible for ACh degradation. BuChE has received
increasing attention in the past years since, as AD progresses,
the activity of AChE decreases, while that of BuChE increases,
in an attempt to modulate ACh levels in cholinergic neurons.
Consequently, both enzymes are therapeutic targets at different
stages of the pathology and, namely, for mild to moderate and
for moderate to severe forms of AD, respectively.5,6
After more than three decades of research efforts in the field,
the treatment of AD still remains an area of significant unmet
need, with therapies based largely on the ChEI, rivastigmine,
donepezil, and galantamine, with the only exception of
memantine, an NMDA (N-methyl-D-aspartate) receptor
antagonist. However, these drugs only target the symptoms
of the disease without altering its progression.
To meet the need of disease-modifying drugs for AD, in
recent years, new approaches have emerged in medicinal
chemistry. In particularm the concept has recently been
proposed that due to the multifactorial and complex etiology
Received: July 28, 2011
Accepted: January 21, 2012
Published: January 21, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
membrane enzyme that catalyzes the hydrolysis of several
endogenous lipid messengers, including eCBs, and therefore, its
inhibitors would allow regulation of eCB signaling and improve
neuronal transmission and/or counteract neuroinflammation,
via CB1 and CB2 receptors, respectively. Accordingly, early
inhibition of eCB inactivation was found to reduce Aβ-induced
gliosis, neuronal death, and memory retention loss.14 Moreover,
recent findings showed that anandamide levels are defective in
cortical areas of postmortem AD brains and are directly
correlated with the positive cognitive scores of the respective
patients and negatively correlated with Aβ42 accumulation.15
Our research groups have been involved for many years in
the development of carbamate ChEIs and various FAAH
inhibitors. Carbamate ChEIs, with the general formula shown
in Figure 1, were designed as potential drug candidates for
and Table 1), coumarin, azaxanthone, and xanthone are
preferred; (b) the optimal chain length (n in the general
formula) proves to be of three methylene units; and (c) the
variation of the length of the carbamic N-substituent has a
remarkable effect on the activity of the inhibitors, with longer
chains yielding more potent inhibitors. Starting from these
results, we have designed four new carbamates (Table 2): 13
and 14 carrying a coumarin core and 15 and 16 with an
azaxanthone one, both substituted with an appropriate bulky
group on the carbamate, in compliance with literature
information on optimal FAAH ligands, but still maintaining
the key features required for ChE inhibition, with the aim of
obtaining multitarget compounds.
According to Scheme 1, compounds 13−16 were synthesized
starting from 7-[N-methyl-N-(3-hydroxybenzyl)amino]-
propoxy-2H-1-benzopyran-2-one17 or 3-[N-methyl-N-(3-hy-
droxybenzyl)-amino]propoxy}-5-azaxanthen-9-one,17 which
were treated with the selected isocyanate in the presence of
NaH.
As reported in Table 2, all new compounds appeared to
inhibit anandamide hydrolysis by FAAH-containing rat brain
membranes. Remarkably, compounds 13 and 15, with the
carbamic N-phenylpentyl substituent, showed activity in the
submicromolar range (IC50 = 0.28 and 0.37 μM, respectively),
which improved to nanomolar (IC50 = 50 and 40 nM) after
preincubation, as expected from pseudoirreversible inhibitors.
When tested on human recombinant FAAH, a remarkable drop
in activity was seen for 13, whereas both 15 and the reference
compound URB597 showed a ∼13-fold decrease in activity.
Inhibitory activities of 13−16 against both cholinesterases
were tested using the method of Ellman24 (Table 2). All
compounds showed a time-dependent pattern of ChEs
inhibition, which is related to the formation of a carbamoylated
covalent adduct with the Ser residue of the enzyme active
site.25,26 Inhibition increased until a steady state, generally
reached within 60−100 and 5−10 min for human AChE and
BuChE, respectively. Thus, IC50 values were determined using
an incubation time suitable for the carbamoylation step to reach
the steady state. Rivastigmine was used as a reference
compound as it is the only marketed carbamate approved for
Figure 1. Lead molecules and general formula of carbamate ChEI.
AD.16−20 The lead xanthostigmine 117 and compound 218
showed the highest activity toward human AChE (IC50 = 0.30
and 0.32 nM).
Taking into account the above-mentioned issues, in view of
the fact that some carbamates are also known from the
literature to be active as FAAH inhibitors21,22 and of the recent
finding that the eCB anandamide and some of its congeners
potently inhibit human plasma BuChE,23 we decided to test the
library16−20of carbamates that was available in house on this
new target.
The inhibitory potencies of compounds showing significant
activity, expressed as IC50 values, against anandamide hydrolysis
by FAAH in rat brain membranes, are reported in Table 1
(inactive compounds not shown). These data highlight some
important structure−activity relationships: (a) among the
different aryl groups (Ar in the general formula of Figure 1
a
Table 1. Inhibitory Activities on FAAH
IC50 FAAH (μM)
Ar
n
R
pit = 0
pit = 20 min
3
4
A
B
3
3
3
3
7
3
3
3
3
3
(CH2)6CH3
(CH2)3CH3
(CH2)2CH3
(CH2)6CH3
CH3
0.62
5.54
9.28
2.21
9.20
4.29
3.62
8.68
6.81
4.23
0.17
0.80
0.10
0.40
NT
0.29
0.11
NT
NT
1.4
5
C
D
E
6
7
8
C
C
A
B
(CH2)5CH3
(CH2)6CH3
(CH2)3CH3
(CH2)6CH3
(CH2)3CH3
9
10
11
12
D
a
pit, preincubation time; NT, not tested; standard error of the mean within 5%.
183
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ACS Medicinal Chemistry Letters
Letter
a
Table 2. Inhibitory Activities of New Designed Compounds against FAAH and Human Cholinesterases
FAAH
b
c
d
IC50 (nM)
IC50 (nM)
IC50 (nM)
Ar
R
pit = 0
pit = 20 min
pit = 20 min
hAChE
74.9
119
89.5
139
hBuChE
1.57
11.2
1.71
27.6
13
14
15
16
U
A
A
D
D
X
Y
X
Y
280
5590
370
50
1820
40
2260
14840
520
4310
2710
6.00
39040
90
R
1535
301
a
b
c
d
pit, preincubation time; U = URB597; R = rivastigmine. Rat brain FAAH. Human recombinant FAAH. Human recombinant AChE and BuChE;
standard error of the mean within 5%.
a
Scheme 1. Synthesis of the Studied Compounds
Information) than the phenylpentyl analogue, in agreement
with previous studies with other morpholino-alkyl carba-
mates.18 Our data also show that independently from the R
substituent, the inhibition kinetics is much faster on hBuChE
than on hAChE (Figure S1 in the Supporting Information).
Compound 13 showed the highest rate of carbamoyl-ChE
formation (k3 = 0.212 min−1 on AChE and 21.4 min−1 on
BuChE), nearly 2 times higher than 14 on AChE and 24 times
higher on BuChE (k3 values for 14, 0.109 min−1 on AChE and
0.893 min−1 on BuChE, Table S1 in the Supporting
Information).
In parallel, the recovery of the enzyme activity after
inhibition followed a similar trend; values of decarbamoylation
constants k5 indicate that velocity of the hydrolysis depends on
the R residue and is higher for the phenylpentyl moiety on both
cholinesterases. For a better understanding of the rate of the
decarbamoylation process, it might be easier to compare the %
of residual activity at a fixed time of dialysis. Indeed, after
inhibition by 13, 83% of initial hBuChE activity and only 30%
of the initial hAChE activity were recovered after 48 h of
dialysis. The k5(13)/k5(14) ratio is 1.87 on hAChE and 5.11 on
hBuChE. These data are in agreement with results obtained by
Perola29 and with our docking studies.18
a
Reagents and conditions: (i) 4-(7-Isocyanatoheptyl)-morpholine or
5-isocyanatopentylbenzene, NaH, room temperature, 24 h.
AD treatment. Results showed that all carbamates acted as
potent butyryl-selective inhibitors with selectivity ranging from
5 (16) to 52.3 (15). Inhibitory potencies were in the low
nanomolar range for hBuChE and in the high nanomolar range
for hAChE inhibition. All compounds were also more potent
cholinesterases inhibitors than the reference compound
rivastigmine. The structural feature mostly affecting the
inhibitory potency on both cholinesterases was the R
substituent at the carbamic nitrogen as compounds 14 and
16, differing at the aryl moiety, have similar inhibition profiles,
while the replacement of the morpholino-heptyl chain of 14
and 16 with the phenyl-pentyl residue (to give 13 and 15,
respectively) increased the inhibitory potency on both AChE
and BuChE, making 13 and 15 the most potent ChEI within
this series.
As previously reported,27 the inhibition of ChEs by
carbamates involves the formation of a reversible complex,
followed by carbamoylation of the enzyme and production of a
covalent adduct (k3, carbamoylation rate constant). The
carbamoylated enzyme is then hydrolyzed by water to
regenerate the free enzyme (k5, decarbamoylation rate
constant). After the reversible complex formation, the
carbamoylation phase of the reaction is considerably more
rapid than the decarbamoylation phase (i.e., k3 ≫ k5), and the
two phases can be characterized separately.28,29 Therefore, we
investigated the rate of inhibition by determining k3, and the
reactivation phase, by determining k5, for two representative
compounds of the new series bearing the phenyl-pentyl (13)
and 7-morpholinoheptyl substituent (14) at the carbamic
nitrogen, respectively, with the final aim to compare the mode
of action of the new inhibitors.
In summary, compound 13, bearing the phenylpentyl
substituent at the N-carbamoyl group, resulted the most potent
and selective BuChE inhibitor in the series, and also one of the
most potent BuChE inhibitors currently known. In the light of
the recent hypothesis regarding a role for BuChE inhibitors in
the treatment of AD, the inhibition profile of 13 might offer
advantages in severe forms of AD. Some general considerations
might be drawn about the physiological relevance of the
kinetics profiles obtained in vitro by comparing data in Table
S1 in the Supporting Information with those obtained for
rivastigmine, a butyryl-selective anti-Alzheimer's drug that acts
as ChEI and is structurally characterized by the presence of a
carbamic function. The interaction of hChE with rivastigmine
was extensively investigated by Bar-On30 and partially re-
evaluated in this study for a better comparison. Specifically, the
k3 value for rivastigmine on hAChE resulted 0.096 min−1, which
makes rivastigmine slightly slower than 13 and as fast as 14.
Noteworthy, compounds 13 and 15 also exhibited high
potency at inhibiting FAAH-catalyzed hydrolysis of ananda-
mide by rat brain membranes. This dual activity renders these
compounds a potentially more efficacious treatment for the
counteraction of AD progression or, at least, of disorders
The data obtained clearly indicate that the nature of the R
substituent at the N-carbamoyl group plays a role in
differentiating both the inhibitory potency and the kinetics of
inhibition. In particular, the 7-morpholinoheptyl derivative 14
carbamoylated and decarbamoylated both cholinesterases more
slowly (lower k3 and k5 values, see Table S1 in the Supporting
184
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ACS Medicinal Chemistry Letters
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ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental procedures of target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +390512099710. Fax: +39 0512099741. E-mail: angela.
rampa@unibo.it (A.R.). Tel: +39081867-5093. Fax:
+390818041770. E-mail: vincenzo.dimarzo@icb.cnr.it (V.D.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Marco Allara
performing the FAAH assays.
̀
for his valuable assistance when
ABBREVIATIONS
■
AD, Alzheimer's disease; Aβ, β-amyloid peptide; AChE,
acetylcholinesterase; BuChE, butyrylcholinesterase; ChEI,
cholinesterase inhibitors; FAAH, fatty acid amide hydrolase;
eCBs, endocannabinoid system
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